This application is a continuation of an ongoing investigation of the mechanisms by which analogs of 1,25-dihydroxyvitamin D3 (1,25D) modulate the transcriptional responses of the vitamin D receptor (VDR). The working hypothesis is that the contact points used by the hormone and analogs in the ligand-binding pocket are different and thereby are able to affect differentially the functional surface of the VDR. Because the surface of the ligand-binding domain of VDR provides an interface for interaction with dimerization partners, transcription coactivators, and corepressors, any subtle change in these interactions may alter the level and spectrum of VDR-mediated gene expression. In Specific Aim 1, Dr. Peleg and her laboratory will complete the analysis of the ligand-binding pocket of the VDR by site-directed mutagenesis. They will define the site of hormone interaction through its 1-alpha-hydroxyl and 25-hydroxyl groups by comparing contact points used by the natural hormone and three types of ligands: 20-epi analogs, analogs with modified A ring and analogs with substitution of their 25-hydroxyl group. In Specific Aim 2, they will determine the effect of differential ligand interaction on the functional surface of the VDR. Again, the use of the natural hormone and two groups of analogs will provide information on the differences and similarities of functional surfaces generated by superagonists (20-epi analogs) and by cell-specific noncalcemic agonists (the A ring-modified analogs). The three types of ligand-receptor complexes will be examined for their potency and efficacy to induce interaction with dimerization partners, coactivators, and corepressors. Using site-directed mutagenesis, the composition of surfaces created by each of these ligands will also be examined. In Specific Aim 3, Dr. Peleg's laboratory will focus on the molecular and cellular mechanism of action of cell-specific analogs. They have identified A ring-modified analogs that have low calcemic activity in vivo, and a profound cell-segregated transcriptional profile in culture. VDR complexes with these analogs will be used as probes to isolate factors that augment or restrict receptor action in a given cellular environment. They will examine whether cell-specific action is due to loss of function by recruitment of a common corepressor, a gain of function due to overexpression of a common coactivator or recruitment of cell-specific factors. These studies will facilitate the development of selective vitamin D receptor modulators that may be useful for treatment of various clinical conditions, including osteoporosis, secondary hyperparathyroidism, and cancer.